b'Sequencing Nucleic acid amplification testsSequencing is a technology that deciphers genetic informationPCR uses sequence-specific primers to amplify a few copies and determines the identity and order of nucleotides in DNA.of DNA to millions, or even billions of copies. When targeting The first milestone was the development of Sanger sequencing,RNA, the RNA is first reverse-transcribed into a complementary a targeted approach with one sequence per reaction. The nextDNA (cDNA) strand prior to PCR amplification. In real-time breakthrough came with the development of NGS technologies.PCR, also known as quantitative PCR (qPCR), an additional NGS covers a variety of different detection chemistries andsequence-specific probe leads to the emission of a fluorescence workflows, each with its advantages and trade-offs. The secondsignal when the target is present and amplified. The fluorescent generation of sequencing undertakes a massively parallelsignal intensity enables a highly accurate quantification of the sequencing approach which involves library preparation andamount of target in a sample relative to a standard with defined target enrichment prior to sequencing. Some popular NGStarget amounts. One hallmark of PCR is that the reaction cycles chemistries are sequencing-by-synthesis, semi-conductorbetween different temperatures. PCR run times vary from sequencing or measurement of electrical current as nucleic acids90120 minutes on average. By utilizing different fluorophores, pass through a nanopore. The turnaround time from sample toqPCR supports multiplex testing that enables the detection of result can vary depending on the platform and application and ismultiple targets in a single reaction. Using the same sample in often between one to several days but can also take more thandifferent qPCR reactions allows for syndromic panel testing.a week. The FDA has approved, cleared, or authorized PCR-based NGS offers comprehensive genomic profiling, pathogenIVDs for a variety of applications, including human genetic, identification, and antibiotic resistance gene detection. NGSmicrobial and companion diagnostic tests. PCR is widely used can be targeted, covering selected fragments of hundredsand accepted for infectious pathogen detection and utilized in of pathogens and/or genetic biomarkers simultaneously.response to COVID-19, including Emergency Use Authorization Alternatively, NGS can be untargeted using whole-exome or(EUA) for at the point-of-care testing. Taking advantage of its whole-genome sequencing. Metagenomic NGS (mNGS) is anmultiplex capability, multiple tests target SARS-CoV-2 and untargeted approach in which all the nucleic acid in a sampleinfluenza A and B in parallel.is sequenced. This provides the most comprehensive geneticDigital PCR (dPCR) allows for the absolute quantification of information of a patient sample and allows the identification anda target without signal-comparison to a standard. Besides characterization of previously unknown emerging pathogens. absolute quantification, dPCR enables the detection of mutations NGS is recognized as an important in vitro diagnostic technology.with very low frequencies or copy number variations (CNVs). In 2018, the Food and Drug Administration (FDA) issued guidanceIsothermal amplification methods are designed to amplify on the development and validation of NGS-based IVDs intendedthe targeted sequence at one constant temperature, which to aid in the diagnosis of suspected germline diseases. Theallows for extremely fast continuous amplification. Isothermal Centers for Medicare & Medicaid Services (CMS) determinedamplification assays can be designed with a visual read-out coverage for patients with somatic cancer in 2018 and forfor easy employment at low-resource settings. In general, patients with germline cancer in 2020. There are currently aisothermal amplification is a promising technology for diagnostic number of FDA-approved IVDs on the market. devices at the point-of-care (POC) and over-the-counter (OTC) Besides cancer, there are many other disease areas that benefitat-home settings. In response to COVID-19, several tests from comprehensive genetic profiles. For example, the Worldbased on isothermal-amplification received EUA, including OTC Health Organization has called out the potential of NGS inat-home tests.diagnosing drug-resistant tuberculosis, but also highlighted challenges such as workflow integration and training, cost and reimbursement, and interpretation of sequencing data. Other areas that benefit from NGS include noninvasive prenatal testing and antibiotic resistance testing in infectious diseases. Sequencing played a key role in the early identification and characterization of SARS-CoV-2 as pathogen causing COVID-19 and in the continuous genomic surveillance of the virus. However, for most routine diagnostics, the cost and turnaround time of sequencing can be prohibitive.5 Molecular testing thermofisher.com/infectiousdisease Contents'